Inhalable formulations for kinase inhibition

ABSTRACT

The invention relates to inhalable formulations configured to inhibit target combinations of kinases for the treatment of cardiovascular and pulmonary diseases such as pulmonary arterial hypertension (PAH).

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of, and priority to, U.S.Provisional Application Nos. 62/849,054, filed May 16, 2019; 62/849,056,filed May 16, 2019; 62/849,058, filed May 16, 2019; 62/849,059, filedMay 16, 2019; 62/877,575, filed Jul. 23, 2019; 62/942,408, filed Dec. 2,2019; 62/984,037, filed Mar. 2, 2020; and 62/958,481, filed Jan. 8,2020; the content of each of which is hereby incorporated by referenceherein in its entirety.

FIELD OF THE INVENTION

The invention generally relates to inhalable formulations of kinaseinhibitors to treat disease.

BACKGROUND

Pulmonary arterial hypertension (PAH) is a condition involving elevatedblood pressure in the arteries of the lungs with unknown causes and isdifferentiated from systemic hypertension. PAH is a progressive diseasewhere resistance to blood flow increases in the lungs causing damage tothe lungs, the pulmonary vasculature and the heart that can eventuallylead to death. While symptoms are treatable with vasodilators and othermedications, there is no known disease modifying therapy or cure andadvanced cases can eventually require lung transplants.

It has been hypothesized that PDGFR plays a significant role in thepathobiology of PAH and that the PDGFR-inhibiting effect of imatinib wastherefore thought to contribute to its efficacy in treating PAH.However, other tyrosine kinase inhibitors active against PDGFR have notshown the same efficacy. In fact, in certain instances PDGFR inhibitorssuch as dasatinib and nintedanib were found to induce or worsen PAH.

Therefore, up to this point, the exact mechanism of action for treatingPAH has remained unknown. Similarly, it is not known what combination ofkinases might be linked to PAH proliferation and might prove usefultreatment targets. Accordingly, an effective treatment for PAH remainselusive.

SUMMARY

The invention is based on an in-depth molecular characterization ofnumerous different compounds. Results of those characterizations haveled to discovery of a molecular profile associated with pulmonaryarterial hypertension (PAH), which profile may be associated with othercondition of the pulmonary cardiovascular system. Particularly, theinvention recognizes that treating conditions such as PAH requiresinhibiting activity of more than just PDGFR, and in fact requiresinhibiting activity of a particular set of kinases.

Accordingly, compositions and methods of the invention target specifickinase combinations for inhibition to treat pulmonary and cardiovasculardiseases such as PAH. By characterizing the binding affinity of imatiniband other PAH-effective compounds against a variety of kinases, newtreatment targets and methods have been identified. Methods of theinvention use the targeted inhibition of kinases such asplatelet-derived growth factor receptors (PDGFRs) and discoidin domainreceptor tyrosine kinase 1 (DDR1), preferably with a single compound, totreat PAH and other pulmonary and cardiovascular diseases.

Methods of the invention may include treating PAH or other pulmonary orcardiovascular diseases through the inhibition of one or more ofPDGFR-β, PDGFR-α, DDR1, colony stimulating factor 1 receptor (CSF1R),tyrosine-protein kinase KIT (KIT), discoidin domain receptor tyrosinekinase 2 (DDR2), lymphocyte-specific protein tyrosine kinase (LCK),Abelson murine leukemia viral oncogene homolog 1 (ABL1), Abelson murineleukemia viral oncogene homolog 2 (ABL2), and phosphatidylinositol5-phosphate 4-kinase type-2 gamma (PI42C). In certain embodiments,pulmonary or cardiovascular diseases such as PAH may be treated throughinhibition of one or more of vascular endothelial growth factor receptor2 (VEGFR-2), HCK proto-oncogene, Src family tyrosine kinase (HCK), fmsrelated receptor tyrosine kinase 4 (FLT4), ret proto-oncogene (RET), SRCproto-oncogene, non-receptor tyrosine kinase (SRC), PDGFR-α, PDGFR-β,DDR1, KIT, CSF1R, fyn related Src family tyrosine kinase (FRK), DDR2,LCK, LYN proto-oncogene, Src family tyrosine kinase (LYN), FYNproto-oncogene, Src family tyrosine kinase (FYN), and FGRproto-oncogene, Src family tyrosine kinase (FGR).

By inhibiting combinations of the above kinases, through theadministration of one or more compounds, methods of the inventionprovide effective treatments for various pulmonary and cardiovasculardiseases such as PAH, pulmonary veno-occlusive disease (PVOD),idiopathic pulmonary fibrosis (IPF), and pulmonary capillaryhemangiomatosis (PCH) as well as lung transplant rejection and pulmonaryhypertension secondary to other diseases like heart failure withpreserved ejection fraction (HFpEF) or schistosomiasis.

Any route of administration may be applicable and certain formulationsand routes of administration are exemplified here. For example, invarious embodiments, compounds are administered in inhalable form as drypowder or through a nebulizer. Inhalable formulations can offer greaterlung exposure than equivalent doses administered through conventionaloral routes or by IV. Accordingly, where a relatively high oral dosewould be required to achieve a target lung exposure, the same exposurecan be achieved with much lower concentrations of drug delivered byinhalation. By avoiding high systemic loads associated with otherconventional administration routes, methods of the invention circumventsome of the adverse effects associated with those high systemicconcentrations. For example, as discussed above, systemicallyadministered imatinib, while proving promising in the treatment of PAH,suffered from unacceptable rates of adverse effects including subduralhematoma. In order to minimize the potential for similar risks based onthe similar kinase-inhibition profiles, certain methods of the inventionprovide inhalable compounds.

Methods of the invention recognize that the inhibition of variouscombinations of specific kinases as discussed above are useful intreating certain cardiovascular and pulmonary ailments. Kinase-bindingcharacterization has been performed for compounds A and B, as detailedbelow, and those compounds have been found to inhibit those variouskinase combinations. Accordingly, in certain embodiments, inhalableformulations of compounds A and/or B may be administered to treatcardiovascular and pulmonary diseases according to the invention.

In certain embodiments, formulations of the invention may be providedwith a higher ratio of API (active pharmaceutical ingredient) than foundin conventional formulations. In certain embodiments, formulationscomprising 50% or more kinase-inhibiting API are provided. Large volumesmay be difficult or dangerous for patients to inhale. Therefore,minimizing the amount of non-API components in the formulation canimprove patient comfort, safety, and compliance by reducing the overallamount of compound that is inhaled while still providing atherapeutically effective API concentration in target tissue.

Furthermore, aerodynamic properties important to inhalable drug uptakecan more easily be managed when less of the formulation is required forcarriers or other additives. By providing functional inhalableformulations with high concentrations of kinase inhibitors or saltsthereof, compositions and methods of the invention can provide theload-reducing benefits discussed above while still deliveringtherapeutic results and avoiding the severe adverse events associatedwith other drug delivery routes.

In various embodiments, the kinase inhibitor(s) or salts thereof used inthe high-API compositions and methods of the invention can consist ofentirely or almost entirely a single crystal form (e.g., greater than80%, 85%, 90%, 95%, 99% or 100% of a single crystal form), therebyallowing for controlled and predictable dosing and patient response. Incertain embodiments, greater than 95% of the kinase inhibiting compound(e.g., compound A or B) or a salt thereof in the inhalable formulationmay be present in a single crystal form.

In certain embodiments inhalable kinase-inhibiting compounds may bemicronized through wet or dry milling (e.g., jet milling) to achieve thedesired particle size for dry powder formulations for inhalation.Compounds or appropriate salts thereof may be micronized to particlesizes of about 0.5 μm to about 5 μm mass median aerodynamic diameter(MMAD) for desired deep lung penetration.

Inhaled products may be limited in terms of the mass of powder that canbe administered and certain salts will contribute significantly to themolecular weight of inhaled formulations. Accordingly, in certainembodiments, the free base of the kinase-inhibiting compound may bepreferred over any salts thereof for efficient delivery of the activemoiety to lung tissue. If required, various excipients or carriers canbe added to the kinase inhibitor(s) or salts thereof before or aftermicronization depending on application. For example, carriers,excipients, conditioners, and force control agents such as lactose(which when used as a carrier may be conditioned with various solventsto increase separation of imatinib during inhalation), magnesiumstearate, leucine, isoleucine, dileucine, trileucine, lecithin,distearylphosphatidylcholine (DSPC) or other lipid-based carriers, orvarious hydrophilic polymers where they exhibit appropriatephysico-chemical properties may be included. The skilled artisan willappreciate that excipients or carriers are optional and that manyembodiments of the invention do not require excipients or carriers. Incompounds including carriers or excipients, API:carrier ratios may begreater than 50:50, 75:25, or 90:10. Additional ratios are contemplatedas discussed below.

In certain embodiments, methods of the invention may includeadministering kinase-inhibiting inhalable formulations that exclude allor most amorphous forms of the compounds. Because crystal form can beimportant to drug pharmacokinetics and dosing, as well asphysicochemical stability and avoiding amorphous content can thereforebe important to providing predictable and efficient therapy.

Treatment methods of the invention may include the administration ofkinase-inhibiting compounds to treat a variety of pulmonary andcardiovascular diseases. Doses may vary depending on the characteristicsof the compound used (e.g., its kinase-binding profile) and the diseasebeing treated. In various embodiments, dose ranges can include betweenabout 10 mg to about 100 mg per dose for inhalation on a twice to fourtimes per day schedule. About 0.1 mg to about 80 mg of the activeimatinib compound may then be deposited within the lungs afterinhalation. The use of relatively high concentrations of API (e.g., 50%or greater) allows for the above doses to be achieved with less overallvolume of inhalable compared to conventional formulations having 1%-3%API.

Methods of the invention may include administration of spray-driedkinase-inhibiting compounds or salts thereof for inhalation. Whilecarriers such as lactose may be used after micronization to aid indelivery via inhalation, those carriers may generally comprise largerdiameter particles and complication in the separation of the activeimatinib compound may result in lower amounts of the inhaled compoundreaching the lungs. Furthermore, the amount of active compound reachingthe lungs may be less predictable using such carriers and methods,making dosing more complicated. Accordingly, spray-dried methods may beused wherein the active kinase-inhibiting compound(s) or salts thereofalong with various excipients or other additives may be micronized to adesired particle size and suspended or solubilized for spray-drying andinhalation.

In certain embodiments, the micronized kinase-inhibiting compound(s) maybe suspended in a feedstock for the purposes of spray-drying to avoidthe creation of amorphous or polymorphic imatinib content that may occurif dissolved in a solution (e.g. in an appropriate organic solvent orwithin an acidified aqueous solution) upon spray-drying. By creating astable suspension of micronized compound for spray-drying, once dried,the inhalable formulation can retain the desired crystal structure,particle size, and low levels of amorphous content obtained before themicronization process.

Stable suspensions for spray-drying may be obtained through manipulationof factors affecting the solubility of the active compound such as pH,ionic strength, and dispersing agents or surfactants. Excipients thatmay be used before micronization in the spray-drying methods describedabove include, for example, leucine, dileucine, trileucine, bulkingagents such as trehalose or mannitol, lecithin, DSPC or otherlipid-based carriers, citrate, or acetate.

Aspects of the invention include methods of treating pulmonary arterialhypertension (PAH) that may comprise providing to a subject atherapeutically effective amount of an inhalable formulation of acompound in order to inhibit activity of a plurality of kinasescomprising one or more platelet-derived growth factor receptors (PDGFRs)and discoidin domain receptor tyrosine kinase 1 (DDR1). The one or morePDGFRs can include PDGFR-β. In certain embodiments, the one or morePDGFRs may include PDGFR-α.

The plurality of kinases can include colony stimulating factor 1receptor (CSF1R). In some embodiments, the plurality of kinases mayinclude tyrosine-protein kinase KIT (KIT). The plurality of kinases caninclude discoidin domain receptor tyrosine kinase 2 (DDR2). Methods ofthe invention may include inhibiting lymphocyte-specific proteintyrosine kinase (LCK).

In certain embodiments, the plurality of inhibited kinases can compriseat least Abelson murine leukemia viral oncogene homolog 1 (ABL1),Abelson murine leukemia viral oncogene homolog 2 (ABL2), colonystimulating factor 1 receptor (CSF1R), discoidin domain receptortyrosine kinase 2 (DDR2), tyrosine-protein kinase KIT (KIT),lymphocyte-specific protein tyrosine kinase (LCK), andphosphatidylinositol 5-phosphate 4-kinase type-2 gamma (PI42C). Each ofthe plurality of kinases may be inhibited with a K_(d) of 500 nM orlower.

Certain aspects of the invention include methods of treating pulmonaryarterial hypertension (PAH) that can include providing to a subject atherapeutically effective amount of an inhalable formulation of acompound in order to inhibit activity of two or more of vascularendothelial growth factor receptor 2 (VEGFR-2), HCK proto-oncogene, Srcfamily tyrosine kinase (HCK), fms related receptor tyrosine kinase 4(FLT4), ret proto-oncogene (RET), SRC proto-oncogene, non-receptortyrosine kinase (SRC), platelet-derived growth factor receptor α(PDGFR-α), platelet-derived growth factor receptor β (PDGFR-β),discoidin domain receptor tyrosine kinase 1 (DDR1), tyrosine-proteinkinase KIT (KIT), colony stimulating factor 1 receptor (CSF1R), fynrelated Src family tyrosine kinase (FRK), discoidin domain receptortyrosine kinase 2 (DDR2), lymphocyte-specific protein tyrosine kinase(LCK), LYN proto-oncogene, Src family tyrosine kinase (LYN), FYNproto-oncogene, Src family tyrosine kinase (FYN), and FGRproto-oncogene, Src family tyrosine kinase (FGR).

DETAILED DESCRIPTION

The invention relates to the treatment of various pulmonary andcardiovascular diseases through the targeted inhibition of specificcombinations of kinases. Diseases including pulmonary arterialhypertension (PAH), pulmonary veno-occlusive disease (PVOD), idiopathicpulmonary fibrosis (IPF), and pulmonary capillary hemangiomatosis (PCH)may be treated as well as lung transplant rejection and pulmonaryhypertension secondary to other diseases like heart failure withpreserved ejection fraction (HFpEF) or schistosomiasis using inhalableformulations of imatinib and salts thereof.

Through comprehensive profiling of the kinase-binding properties ofvarious compounds thought to effectively treat cardiovascular andpulmonary diseases like PAH, specific combinations of kinases have beenidentified as targets for inhibition. By treating patients withinhalable compounds that inhibit those target kinases, methods of theinvention can provide effective treatment of many such diseases.

In certain embodiments, methods of the invention can include targetedinhibition of combinations of Abelson murine leukemia viral oncogenehomolog 1 (ABL1), colony stimulating factor 1 receptor (CSF1R),discoidin domain receptor tyrosine kinase 1 (DDR1), discoidin domainreceptor tyrosine kinase 2 (DDR2), tyrosine-protein kinase KIT (KIT),lymphocyte-specific protein tyrosine kinase (LCK), platelet-derivedgrowth factor receptor-α (PDGFR-α), and platelet-derived growth factorreceptor-β (PDGFR-β). The above kinases were found to be inhibited byboth compound A and compound B.

In certain embodiments, methods of the invention can include targetedinhibition of combinations of PDGFR-β, PDGFR-α, DDR1, CSF1R, KIT, DDR2,LCK, ABL1, Abelson murine leukemia viral oncogene homolog 2 (ABL2), andphosphatidylinositol 5-phosphate 4-kinase type-2 gamma (PI42C). Theabove kinases were found to be inhibited by compound A.

In certain embodiments, methods of the invention can include targetedinhibition of combinations of vascular endothelial growth factorreceptor 2 (VEGFR-2), HCK proto-oncogene, Src family tyrosine kinase(HCK), fms related receptor tyrosine kinase 4 (FLT4), ret proto-oncogene(RET), SRC proto-oncogene, non-receptor tyrosine kinase (SRC), PDGFR-α,PDGFR-β, DDR1, KIT, CSF1R, fyn related Src family tyrosine kinase (FRK),DDR2, LCK, LYN proto-oncogene, Src family tyrosine kinase (LYN), FYNproto-oncogene, Src family tyrosine kinase (FYN), and FGRproto-oncogene, Src family tyrosine kinase (FGR). The above kinases werefound to be inhibited by compound B

In certain embodiments, methods of the invention may include targetedinhibition of combinations of PDGFR-β, PDGFR-α, DDR1, CSF1R, KIT, DDR2,LCK, ABL1, ABL2, and PI42C while not significantly inhibiting (e.g. lessthan 500 nM K_(d)) one or more of VEGFR-2, HCK, FLT4, RET, SRC, FRK,LYN, FYN, and FGR.

In certain embodiments, methods of the invention may include targetedinhibition of combinations of VEGFR-2, HCK, FLT4, RET, SRC, PDGFR-α,PDGFR-β, DDR1, KIT, CSF1R, FRK, DDR2, LCK, LYN, FYN, and FGR while notsignificantly inhibiting (e.g. less than 500 nM K_(d)) one or more ofABL2, and PI42C.

In various embodiments, methods of the invention may include targetedinhibition of any combination of the above kinases via administration ofa compound having an equilibrium dissociation constant (K_(d)) of lessthan about 1000 nM, 500 nM, less than about 400 nM, less than about 300nM, less than about 200 nM, less than about 100 nM, less than about 75nM, less than about 50 nM, less than about 40 nM, less than about 30 nM,less than about 20 nM, less than about 15 nM, or less than about 5 nMwith respect to each of the targeted kinases.

Compound A, as characterized below, is free base imatinib having thefollowing structure:

The kinase-binding profile including the equilibrium dissociationconstant (K_(d)) was determined for compound A as detailed in Example 1below. The results of that characterization are shown in Table 1.

TABLE 1 Compound Activity Kinase Remaining at 1 μM (%) K_(d) (nM) KIT0.5 13 PDGFR-β 0.15 14 DDR1 0.2 0.7 VEGFR2 70 >10000 PDGFR-α 0.95 31CSF1R 1.6 11 LCK 1.2 40 DDR2 0.8 15 ABL-1-nonphosphorolated 0.05 1.1 HCK81 >10000 PIK4CB 59 >10000 RET 98 >10000 FRK 56 1500 SRC 94 >10000 LYN30 890 FYN 79 3100 FLT4 87 >10000 FGR 70 2400

Compound B, as characterized below, has the following structure:

Compound B and some of its potential applications in treating pulmonaryand cardiovascular disorders are discussed in U.S. Pat. Nos. 9,815,815;9,925,184; 10,231,966; and 10,246,438; the content of each of which isincorporated herein by reference.

The kinase-binding profile including the equilibrium dissociationconstant (K_(d)) was determined for compound B as detailed in Example 3below. The results of that characterization are shown in Table 2.

TABLE 2 Compound Activity Kinase Remaining at 1 μM (%) K_(d) (nM) KIT 04.7 PDGFR-β 0 0.82 DDR1 0.2 3.5 VEGFR2 0.25 15 PDGFR-α 0.35 4 CSF1R 0.76.1 LCK 1.1 22 DDR2 1.8 19 ABL-1-nonphosphorolated 3.3 — HCK 3.3 36PIK4CB 3.8 150 RET 3.9 19 FRK 5.3 11 SRC 6.4 130 LYN 7.3 24 FYN 9.7 55FLT4 12 18 FGR 21 92

As shown in the tables above, both compounds significantly bind andtherefore inhibit a number of the same kinases while each furtherinhibits additional kinases not significantly affected by the othercompound. By analyzing those combined profiles, in certain embodiments,methods of the invention are able to identify target kinases forinhibition that are common to both compounds and that, therefore may bemore likely to provide a therapeutic effect in treating cardiovascularor pulmonary diseases.

Furthermore, the differences in kinase-inhibition between the twocompounds may account for differences in compound efficacy in treatingcertain pulmonary and cardiovascular diseases and account for variousadverse effects. Accordingly, in certain embodiments, methods of theinvention may target only kinases inhibited by one or the other of thecompounds to improve treatment outcomes and reduce the risk of adverseeffects.

In certain embodiments methods and compositions described herein mayprovide greater concentrations of a kinase-inhibiting compound in targetlung tissue than obtained with equivalent doses administered orally orthrough IV. Furthermore, those doses, comprising a high percentage ofthe overall formulation, can be delivered in lower volume formulationsthan conventional formulations of between 1% and 3% API. Reducing thevolume a patient must inhale can increase patient comfort andcompliance, thereby improving results. Additionally, a higher percentageof API content can improve the API distribution and blend uniformity.Accordingly, methods and compositions of the invention allow fortreatment of conditions of the pulmonary cardiovascular system (e.g.,PAH) with lower doses and less inhalable volume than would be requiredin systemic administration, thereby lowering the risk of adverse eventsincluding subdural hematoma (See, Frost et al.). Thus, the inventionprovides viable treatment methods for life threatening diseases thatwere heretofore too risky for practical application.

In certain embodiments, compounds of the invention may includeformulations of a kinase inhibitor such as imatinib or salts thereoftargeting combinations of kinases as discussed above. In certainembodiments, the free base of the kinase-inhibiting compound may be usedin a formulation (either in dry powder or suspension) for inhalation totreat a condition of the pulmonary cardiovascular system such as PAH.Certain salt forms are also contemplated. In various embodiments, kinaseinhibitor salts that were found to exhibit suitable thermal stabilityand few or single polymorphic forms include glycollate, isethionate,malonate, tartrate, and malate. Other salt forms contemplated herein arexinafoate, furoate, trifenatate, HCl, sulfate, phosphate, lactate,maleate, fumarate, succinate, adipate, mesylate, and citrate.

When the compounds of the present invention are administered aspharmaceuticals, to humans and mammals, they can be given alone or as apharmaceutical composition containing, for example, 0.1 to 99.5% ofactive ingredient (e.g., imatinib or a salt thereof) in combination witha pharmaceutically acceptable carrier. In preferred embodiments, toreduce inhaled volumes for patients and improve patient outcomes,formulations can comprise at least 50% of a kinase-inhibiting compoundor a salt thereof.

In certain embodiments, formulations of the invention may include one ormore excipients. Excipients may include, for example, lactose in variousforms (e.g., roller dried or spray dried). Larger lactose particles canbe used as a carrier for inhalation of micronized formulations. Thecarrier particles, with their larger size, can be used to increaseaerodynamic forces on the combined kinase inhibitor/carrier in order toaid in delivery through inhalation. Solvents may be used to conditionthe lactose surface such that the active component can be effectivelyseparated from the lactose as it leaves the inhaler device and withinthe oral cavity when being used as a carrier. Magnesium stearate can beused as a force-control agent or conditioning agent in variousembodiments. In some embodiments, leucine can be used as a force-controlagent including different forms of leucine (e.g. isoleucine) along withdileucine and even trileucine.

Lecithin phospholipids such as DSPC may be used as an excipient for drypowder inhalation. In certain embodiments, excipients may includevarious hydrophilic polymers. See, for example, Karolewicz, B., 2016, Areview of polymers as multifunctional excipients in drug dosage formtechnology, Saudi Pharm J., 24(5):525-536, incorporated herein byreference.

In the high-API-ratio formulations contemplated herein, carriers orexcipients may make up the remainder of the formulation in amounts of50% or less of the overall composition. In certain embodiments,inhalable formulations may have API:carrier ratios of 50:50, 55:45,60:40, 65:35, 70:30, 75:25, 80:20, 85:15, 90:10, or 95:5. Certaininhalable formulations may be pure API with no additional components. Invarious embodiments, formulations may include a kinase inhibitor orsalts thereof as the API in amounts greater than 5%, 10%, 15%, 20%, 25%,30%, 35%, 40%, or 45%. As used herein, API ratios refer to % w/w.

In various embodiments, micronized kinase inhibitor and salts thereofretain crystallinity, even after micronization and spray drying (asdiscussed in detail below). For example, kinase inhibiting formulationsof the invention can include less than 50%, less than 25%, less than20%, less than 10%, less than 9%, less than 8%, less than 7%, less than6%, less than 5%, less than 4%, less than 3%, less than 2%, or less than1% amorphous API content by mass. In preferred embodiments, formulationsof the invention include no observable amorphous content of thekinase-inhibiting compound. Of particular note is, by suspendingmicronized kinase inhibitor particles in a solution as opposed tosolubilizing, the desired crystalline form and low amorphous contentobtained during micronization is carried through to the spray-driedinhalable powder because the kinase inhibitor crystals may not bedissolved in the solution to a significant degree.

Another unexpected result obtained with methods and formulations of theinvention is that kinase inhibitor formulations of the invention may besignificantly less hygroscopic than conventional salt compounds such asimatinib mesylate. Accordingly, the formulations of the invention arebetter suited for dry powder inhalation and can comprise less than 5%water content, less than 4%, less than 3%, less than 2%, or, inpreferred embodiments, less than 1% water content.

As discussed above, in order to accurately and consistently modelpharmacokinetics of formulations for proper dosing, low polymorphism isdesired. To that end, inhalable formulations of the invention includekinase inhibitors or salts thereof present in a single crystal form. Invarious embodiments, the kinase inhibitor or a salt thereof may bepresent at greater than 75%, 80%, 85%, 90%, 95%, or, in preferredembodiments, greater than 99% in a single crystal form by mass. Thesingle crystal form may be, for example, imatinib in type A or type B invarious embodiments. Crystalline purity can be estimated using any knownmethod including, for example, x-ray powder diffraction (XRPD).

In various embodiments, kinase inhibitors or salts thereof are providedin dry powder formulations for inhalation. Dry powder can beadministered via, for example, dry powder inhalers such as described inBerkenfeld, et al., 2015, Devices for Dry Powder Drug Delivery to theLung, AAPS PharmaSciTech, 16(3):479-490, incorporated herein byreference. Dry powder compounds may be divided into single doses forsingle, twice daily, three times daily, or four times daily inhalationto treat disorders such as PAH or other conditions of the pulmonarycardiovascular system. The single doses may be divided into individualcapsules or other formats compatible with the dry powder inhaler to beused.

In other embodiments, suspensions having the characteristics describedherein (e.g., low polymorphism and amorphous content) can be deliveredvia inhalation using, for example, a nebulizer. Suspensions may offeradvantages over solutions as discussed below. For nebulized suspensions,micronization and particle diameter may be of particular importance forefficient delivery and the active kinase-inhibiting compound may bepreferably micronized to a mass median diameter of 2 μm or less. Thesuspension solution for nebulizer inhalation can be aqueous and dosesmay be divided into individual containers or compartments for sterilestorage prior to use.

Micronized kinase inhibitor particle size can range from about 0.5 μm toabout 5 μm depending on application (e.g., dry powder or suspension forinhalation). In preferred embodiments the size range is about 1 μm toabout 3 μm in dry powder formulations to achieve deep lung penetration.

Dosages for treating PAH and other conditions of the pulmonarycardiovascular system may be in the range of between about 10 mg toabout 100 mg per dose for inhalation on once, twice or three times perday schedule. About 0.1 mg to about 80 mg of the kinase inhibitor(s) orsalts thereof may then be deposited within the lung after inhalation. Incertain embodiments about 10 mg to 30 mg of kinase-inhibiting compoundmay be given in a capsule for a single dry-powder inhalation dose withabout 5 mg to about 10 mg of the compound to be expected to reach thelungs. In inhalable suspension embodiments, kinase inhibitor may bepresent at about 0.1 to about 1 mg/kg in a dose and may be administeredone to four times a day to obtain the desired therapeutic results.

In certain embodiments, the kinase-inhibiting methods of the inventionmay be used to treat pulmonary hypertension as a result ofschistosomiasis. See, for example, Li, et al., 2019, The ABL kinaseinhibitor imatinib causes phenotypic changes and lethality in adultSchistosoma japonicum, Parasitol Res., 118(3):881-890; Graham, et al.,2010, Schistosomiasis-associated pulmonary hypertension: pulmonaryvascular disease: the global perspective, Chest, 137(6 Suppl):20S-29S,the content of each of which is incorporated herein by reference.

Methods and compositions of the invention may be used to treat lungtransplant recipients to prevent organ rejection. See, Keil, et al.,2019, Synergism of imatinib, vatalanib and everolimus in the preventionof chronic lung allograft rejection after lung transplantation (LTx) inrats, Histol Histopathol, 1:18088, incorporated herein by reference.

In certain embodiments, pharmaceutical compositions described herein canbe used to treat pulmonary veno-occlusive disease (PVOD). See Sato, etal., 2019, Beneficial Effects of Imatinib in a Patient with SuspectedPulmonary Veno-Occlusive Disease, Tohoku J Exp Med. 2019 February;247(2):69-73, incorporated herein by reference.

For treatment of any conditions of the pulmonary cardiovascular systemfor which kinase-inhibiting methods of the invention may produce atherapeutic effect, compounds and methods of the invention may be usedto provide greater concentration at the target lung tissue throughinhalation along with consistent, predictable pharmacokinetics affordedby low polymorphism and amorphous content. The efficient localization oftherapeutic compound at the target tissue allows for lower systemicexposure and avoidance of the adverse events associated with prolongedoral administration of certain kinase inhibitors such as imatinibmesylate.

Methods of the invention can include preparation of kinase-inhibitingformulations. As noted above, kinase inhibitors or salts thereof may beadministered via inhalation in suspension or dry powder form. Dry powderformulations may be obtained via any known method including, inpreferred embodiments, jet milling. Jet milling can be used to grindactive compounds and, potentially, various additives (e.g., excipients)using a jet (or jets) of compressed air or gas to force collisionsbetween the particles as they transit at near sonic velocity around theperimeter of a toroidal chamber. The size reduction is the result of thehigh-velocity collisions between particles of the process material.Outputs of the jet mill may allow particles to exit the apparatus once adesired size has been reached. As noted herein, desired particle sizefor dry powder inhalation and other formulations may be in the range ofabout 0.5 μm to about 5 μm.

In certain embodiments, bulk compounds may be micronized to the desiredsize for inhalation via wet milling wherein the kinase inhibitorparticles are suspended in a slurry and reduced through shearing orimpact with a grinding media.

An unexpected finding of the invention is that, once micronized, freebase kinase-inhibiting compounds retain crystallinity and areconsiderably less hygroscopic than certain salt forms (e.g., imatinibmesylate). Furthermore, micronized imatinib obtained using methods ofthe invention has been found to exhibit no apparent polymorphs otherthan the designated Type A and very low levels of amorphous content.Accordingly, this can result in improved stability of the drug substanceand any drug product upon storage. Single crystal forms of imatinib orother kinase inhibitors such as described may allow for more predictablein vivo behavior and appropriate dosing can be determined.

Once micronized, in dry powder form, kinase inhibitor formulations ofthe invention, with their low polymorphic and amorphous content, can beprepared for inhalation. In certain embodiments, the dry powder kinaseinhibitor can be combined with larger carrier particles such as lactoseas discussed above.

In some embodiments a suspension can be formed of the kinase inhibitorcompound(s). The suspension may result from dry micronization followedby suspension of the resulting dry powder or can be obtained as theoutcome of a wet milling procedure. Suspensions of micronized crystalforms may be used in nebulized inhalation treatment or may be spraydried for dry powder treatments.

Spray drying methods may follow the following procedure. First, bulkkinase inhibitor compound may be micronized as described above to obtainparticles in a desired size range. Then the micronized compound can besuspended in a solution such that it does not dissolve and insteadretains the desired crystalline features (e.g., low polymorphism andamorphous content). The suspended particles can then be spray driedusing any known method. Spray drying techniques are well characterizedand described, for example, in Ziaee, et al., 2019, Spray drying ofpharmaceuticals and biopharmaceuticals: Critical parameters andexperimental process optimization approaches, Eur. J. Pharm. Sci.,127:300-318, and Weers et al., 2019, AAPS PharmSciTech. 2019 Feb. 7;20(3):103. doi: 10.1208/s12249-018-1280-0, and 2018/0303753, each ofwhich is incorporated herein by reference. Spray drying micronizedkinase inhibitor compounds or salts thereof provides for uniform andpredictable crystallinity and particle size and can avoid the need forlarge carrier molecules that may adversely affect the amount of inhaleddrug that reaches the target lung tissue.

In spray-dried embodiments, micronized drug particles may be suspendedwithin a non-aqueous solvent or within an emulsion of a non-aqueoussolvent which, in turn is emulsified or dispersed within an aqueousenvironment (e.g. oil in water) and spray-dried, resulting incrystalline drug particles. The non-aqueous component may or may not befugitive and thus could be removed completely during spray drying or, itcould be retained, depending on the desired properties required. In suchembodiments, each atomized droplet (mass median diameter ˜10 μm)contains dispersed drug crystals. During the initial moments of thedrying process, the more volatile aqueous phase begins to evaporate. Therapidly receding atomized droplet interface drives enrichment of theslowly diffusing drug and emulsion particles at the interface. Thisleads to formation of a void space in the center of the drying droplet.As the drying process continues, the less volatile oil phase in theemulsion droplets evaporates, resulting in formation of hollow pores intheir place. Overall, the resulting hollow spray-dried compositeparticles contain drug crystals.

As maintaining a stable solution of crystalline kinase inhibitorcompound is important to many features of the formulations and methodsof the invention, formulation methods include manipulation of thesuspension to prevent dissolution of the kinase inhibitor compound.Aqueous solution factors such as pH, ionic strength and dispersingagents may be used to obtain a stable suspension for nebulizedinhalation or spray drying. For example, the pH of the aqueous solutionmay be adjusted to prevent dissolution.

Additionally, the presence of ions in aqueous solution may tend to ‘saltout’ the active compound. The solubility of the kinase inhibitors andany salts thereof may decrease with salinity. Accordingly, salt in theaqueous solution may be used to reduce solubility of the active compoundcrystals in certain embodiments.

To promote dispersion and thoroughly deagglomerate the API particles, adispersing agent or surfactant (e.g., Tween 20 or Tween 80) may be addedbut should not cause dissolution of the kinase inhibitor in suspension.

In certain embodiments, excipients can be added to the suspension beforespray drying. In various embodiments, the excipient may be awater-soluble excipient, such as leucine, dileucine, trileucine,trehalose, mannitol, citrate or acetate. In other embodiment, theexcipient may be a water insoluble excipient, such as lecithin,distearylphosphatidylcholine (DSPC) or limonene. Such insolubleexcipients may be dissolved in a non-aqueous medium that is miscible orimmiscible with water, thereby creating an emulsion. Alternatively, aliposomal dispersion could be created into which the suspended kinaseinhibitor could be added and homogenized or where it could be spraydried in separate feedstocks.

The effective dosage of each agent can readily be determined by theskilled person, having regard to typical factors such as the age,weight, sex and clinical history of the patient. In general, a suitabledaily dose of a compound of the invention will be that amount of thecompound which is the lowest dose effective to produce the desiredtherapeutic effect. Such an effective dose will generally depend uponthe factors described above.

If desired, the effective daily dose of the active compound may beadministered as one, two, three, four, five, six or more sub-dosesadministered separately at appropriate intervals throughout the day,optionally, in unit dosage forms.

The pharmaceutical compositions of the invention include a“therapeutically effective amount” or a “prophylactically effectiveamount” of one or more of the compounds of the present invention, orfunctional derivatives thereof. An “effective amount” refers to anamount effective, at dosages and for periods of time necessary, toachieve the desired therapeutic result, e.g., a diminishment orprevention of effects associated with PAH. A therapeutically effectiveamount of a compound of the present invention or functional derivativesthereof may vary according to factors such as the disease state, age,sex, and weight of the subject, and the ability of the therapeuticcompound to elicit a desired response in the subject. A therapeuticallyeffective amount is also one in which any toxic or detrimental effectsof the therapeutic agent are outweighed by the therapeuticallybeneficial effects.

A “prophylactically effective amount” refers to an amount effective, atdosages and for periods of time necessary, to achieve the desiredprophylactic result. Typically, since a prophylactic dose is used insubjects prior to, or at an earlier stage of disease, theprophylactically effective amount may be less than the therapeuticallyeffective amount. A prophylactically or therapeutically effective amountis also one in which any toxic or detrimental effects of the compoundare outweighed by the beneficial effects.

Dosage regimens may be adjusted to provide the optimum desired response(e.g. a therapeutic or prophylactic response). For example, a singleinhalable bolus may be administered, several divided doses may beadministered over time or the dose may be proportionally reduced orincreased as indicated by the exigency of the therapeutic situation.Actual dosage levels of the active ingredients in the pharmaceuticalcompositions of this invention may be varied so as to obtain an amountof the active ingredient which is effective to achieve the desiredtherapeutic response for a particular subject, composition, and mode ofadministration, without being toxic to the patient.

The term “dosage unit” as used herein refers to physically discreteunits suited as unitary dosages for the mammalian subjects to betreated; each unit containing a predetermined quantity of activecompound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on (a) the unique characteristics of the compound, and (b) thelimitations inherent in the art of compounding such an active compoundfor the treatment of sensitivity in individuals.

In some embodiments, therapeutically effective amount can be estimatedinitially either in cell culture assays or in animal models, usuallyrats, non-human primates, mice, rabbits, dogs, or pigs. The animal modelis also used to achieve a desirable concentration range and route ofadministration. Such information can then be used to determine usefuldoses and routes for administration in other subjects. Generally, thetherapeutically effective amount is sufficient to reduce PAH symptoms ina subject. In some embodiments, the therapeutically effective amount issufficient to eliminate PAH symptoms in a subject.

Dosages for a particular patient can be determined by one of ordinaryskill in the art using conventional considerations, (e.g. by means of anappropriate, conventional pharmacological protocol). A physician may,for example, prescribe a relatively low dose at first, subsequentlyincreasing the dose until an appropriate response is obtained. The doseadministered to a patient is sufficient to effect a beneficialtherapeutic response in the patient over time, or, e.g., to reducesymptoms, or other appropriate activity, depending on the application.The dose is determined by the efficacy of the particular formulation,and the activity, stability, or half-life of the compounds of theinvention or functional derivatives thereof, and the condition of thepatient, as well as the body weight or surface area of the patient to betreated. The size of the dose is also determined by the existence,nature, and extent of any adverse side-effects that accompany theadministration of a particular vector, formulation, or the like in aparticular subject. Therapeutic compositions comprising one or morecompounds of the invention or functional derivatives thereof areoptionally tested in one or more appropriate in vitro and/or in vivoanimal models of disease, such as models of PAH, to confirm efficacy,tissue metabolism, and to estimate dosages, according to methods wellknown in the art. In particular, dosages can be initially determined byactivity, stability or other suitable measures of treatment vs.non-treatment (e.g., comparison of treated vs. untreated cells or animalmodels), in a relevant assay. Administration can be accomplished viasingle or divided doses.

In certain embodiments, in which an aqueous suspension is part of themanufacturing process, the aqueous suspension may contain the activematerial in admixture with excipients suitable for the manufacture ofaqueous suspensions. Such excipients are suspending agents dispersing orwetting agents such as a naturally occurring phosphatide, for examplelecithin, or condensation products of an alkylene oxide with fattyacids, for example polyoxyethylene stearate, or condensation products ofethylene oxide with long chain aliphatic alcohols, for exampleheptadecaethyleneoxycetanol, or condensation products of ethylene oxidewith partial esters derived from fatty acids and a hexitol such apolyoxyethylene with partial esters derived from fatty acids and hexitolanhydrides, for example polyoxyethylene sorbitan monooleate. The aqueoussuspensions may also contain one or more preservatives, for exampleethyl, or n-propyl p-hydroxybenzoate, one or more coloring agents, oneor more flavoring agents, and one or more sweetening agents, such assucrose, mannitol, or trehalose.

Dispersible powders and granules suitable for preparation of an aqueoussuspension by the addition of water provide the active ingredient inadmixture with a dispersing or wetting agent, suspending agent and oneor more preservatives.

The term “pharmaceutical composition” means a composition comprising acompound as described herein and at least one component comprisingpharmaceutically acceptable carriers, diluents, adjuvants, excipients,or vehicles, such as preserving agents, taste-masking agents, fillers,disintegrating agents, wetting agents, emulsifying agents, suspendingagents, sweetening agents, flavoring agents, perfuming agents,antibacterial agents, antifungal agents, lubricating agents anddispensing agents, depending on the nature of the mode of administrationand dosage forms.

The term “pharmaceutically acceptable carrier” is used to mean anycarrier, diluent, adjuvant, excipient, or vehicle, as described herein.Examples of suspending agents include ethoxylated isostearyl alcohols,polyoxyethylene sorbitol and sorbitan esters, microcrystallinecellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth,or mixtures of these substances. Prevention of the action ofmicroorganisms can be ensured by various antibacterial and antifungalagents, for example, parabens, chlorobutanol, phenol, sorbic acid, andthe like. It may also be desirable to include isotonic agents, forexample sugars, sodium chloride, and the like. Examples of suitablecarriers, diluents, solvents, or vehicles include water, ethanol,polyols, suitable mixtures thereof, vegetable oils (such as olive oil),and organic esters such as ethyl oleate. Examples of excipients includelactose, milk sugar, sodium citrate, calcium carbonate, and dicalciumphosphate. Examples of disintegrating agents include starch, alginicacids, and certain complex silicates. Examples of lubricants includemagnesium stearate, sodium lauryl sulphate, talc, as well as highmolecular weight polyethylene glycols.

The term “pharmaceutically acceptable” means it is, within the scope ofsound medical judgment, suitable for use in contact with the cells ofhumans and lower animals without undue toxicity, irritation, allergicresponse, and the like, and are commensurate with a reasonablebenefit/risk ratio.

INCORPORATION BY REFERENCE

References and citations to other documents, such as patents, patentapplications, patent publications, journals, books, papers, webcontents, have been made throughout this disclosure. All such documentsare hereby incorporated herein by reference in their entirety for allpurposes.

Equivalents

Various modifications of the invention and many further embodimentsthereof, in addition to those shown and described herein, will becomeapparent to those skilled in the art from the full contents of thisdocument, including references to the scientific and patent literaturecited herein. The subject matter herein contains important information,exemplification and guidance that can be adapted to the practice of thisinvention in its various embodiments and equivalents thereof.

EXAMPLES Example 1

An inhalable form of imatinib (compound A) was screened against 468quantifiable kinases in the human kinome (the set of protein kinasesencoded in the human genome) to characterize binding affinity for eachkinase.

Kinase binding was profiled using KINOMEscan® (Eurofins DiscoverXCorporation). KINOMEscan™ is based on a competition binding assay thatquantitatively measures the ability of a compound to compete with animmobilized, active-site directed ligand. The assay is performed bycombining three components: DNA-tagged kinase; immobilized ligand; and atest compound. The ability of the test compound to compete with theimmobilized ligand is measured via quantitative PCR of the DNA tag.

Kinase-tagged T7 phage strains were prepared in an E. coli host derivedfrom the BL21 strain. E. coli were grown to log-phase and infected withT7 phage and incubated with shaking at 32° C. until lysis. The lysateswere centrifuged and filtered to remove cell debris. Some kinases wereproduced in HEK-293 cells and subsequently tagged with DNA for qPCRdetection.

Streptavidin-coated magnetic beads were treated with biotinylated smallmolecule ligands for 30 minutes at room temperature to generate affinityresins for kinase assays. The ligand-bound beads were blocked withexcess biotin and washed with blocking buffer (SeaBlock (Pierce), 1%BSA, 0.05% Tween 20, 1 mM DTT) to remove unbound ligand and to reducenonspecific binding. Binding reactions were assembled by combiningkinases, ligand-bound affinity beads, and test compounds in 1× bindingbuffer (20% SeaBlock, 0.17×PBS, 0.05% Tween 20, 6 mM DTT). Testcompounds were prepared as 111× stocks in 100% DMSO. K_(d)s weredetermined using an 11-point 3-fold compound dilution series with threeDMSO control points. All compounds for K_(d) measurements weredistributed by acoustic transfer (non-contact dispensing) in 100% DMSO.

The compounds were then diluted directly into the assays such that thefinal concentration of DMSO was 0.9%. All reactions were performed inpolypropylene 384-well plates. Each was a final volume of 0.02 ml. Theassay plates were incubated at room temperature with shaking for 1 hourand the affinity beads were washed with wash buffer (1×PBS, 0.05% Tween20). The beads were then re-suspended in elution buffer (1×PBS, 0.05%Tween 20, 0.5 μM non-biotinylated affinity ligand) and incubated at roomtemperature with shaking for 30 minutes. The kinase concentration in theeluates was measured by qPCR.

Example 2

Compound B, as detailed above, was screened against 468 quantifiablekinases in the human kinome (the set of protein kinases encoded in thehuman genome) to characterize binding affinity for each kinase.

Kinase binding was profiled using KINOMEscan® (Eurofins DiscoverXCorporation). KINOMEscan™ is based on a competition binding assay thatquantitatively measures the ability of a compound to compete with animmobilized, active-site directed ligand. The assay is performed bycombining three components: DNA-tagged kinase; immobilized ligand; and atest compound. The ability of the test compound to compete with theimmobilized ligand is measured via quantitative PCR of the DNA tag.

Kinase-tagged T7 phage strains were prepared in an E. coli host derivedfrom the BL21 strain. E. coli were grown to log-phase and infected withT7 phage and incubated with shaking at 32° C. until lysis. The lysateswere centrifuged and filtered to remove cell debris. Some kinases wereproduced in HEK-293 cells and subsequently tagged with DNA for qPCRdetection.

Streptavidin-coated magnetic beads were treated with biotinylated smallmolecule ligands for 30 minutes at room temperature to generate affinityresins for kinase assays. The ligand-bound beads were blocked withexcess biotin and washed with blocking buffer (SeaBlock (Pierce), 1%BSA, 0.05% Tween 20, 1 mM DTT) to remove unbound ligand and to reducenonspecific binding. Binding reactions were assembled by combiningkinases, ligand-bound affinity beads, and test compounds in 1× bindingbuffer (20% SeaBlock, 0.17×PBS, 0.05% Tween 20, 6 mM DTT). Testcompounds were prepared as 111× stocks in 100% DMSO. K_(d)s weredetermined using an 11-point 3-fold compound dilution series with threeDMSO control points. All compounds for K_(d) measurements weredistributed by acoustic transfer (non-contact dispensing) in 100% DMSO.

The compounds were then diluted directly into the assays such that thefinal concentration of DMSO was 0.9%. All reactions were performed inpolypropylene 384-well plates. Each was a final volume of 0.02 ml. Theassay plates were incubated at room temperature with shaking for 1 hourand the affinity beads were washed with wash buffer (1×PBS, 0.05% Tween20). The beads were then re-suspended in elution buffer (1×PBS, 0.05%Tween 20, 0.5 μM non-biotinylated affinity ligand) and incubated at roomtemperature with shaking for 30 minutes. The kinase concentration in theeluates was measured by qPCR.

What is claimed is:
 1. A method of treating pulmonary arterialhypertension (PAH), the method comprising providing to a subject atherapeutically effective amount of an inhalable formulation of acompound in order to inhibit activity of a plurality of kinasescomprising one or more platelet-derived growth factor receptors (PDGFRs)and discoidin domain receptor tyrosine kinase 1 (DDR1).
 2. The method ofclaim 1 wherein the one or more PDGFRs comprises PDGFR-β.
 3. The methodof claim 2 wherein the one or more PDGFRs comprises PDGFR-α.
 4. Themethod of claim 3 wherein the plurality of kinases comprises colonystimulating factor 1 receptor (CSF1R).
 5. The method of claim 4 whereinthe plurality of kinases comprises tyrosine-protein kinase KIT (KIT). 6.The method of claim 5 wherein the plurality of kinases comprisesdiscoidin domain receptor tyrosine kinase 2 (DDR2).
 7. The method ofclaim 6 wherein the plurality of kinases comprises lymphocyte-specificprotein tyrosine kinase (LCK).
 8. The method of claim 1 wherein theplurality of kinases comprise Abelson murine leukemia viral oncogenehomolog 1 (ABL1), Abelson murine leukemia viral oncogene homolog 2(ABL2), colony stimulating factor 1 receptor (CSF1R), discoidin domainreceptor tyrosine kinase 2 (DDR2), tyrosine-protein kinase KIT (KIT),lymphocyte-specific protein tyrosine kinase (LCK), andphosphatidylinositol 5-phosphate 4-kinase type-2 gamma (PI42C).
 9. Themethod of claim 8 wherein each of the plurality of kinases are inhibitedwith a K_(d) of 500 nM or lower.
 10. A method of treating pulmonaryarterial hypertension (PAH), the method comprising providing to asubject a therapeutically effective amount of an inhalable formulationof a compound in order to inhibit activity of a plurality of kinasescomprising vascular endothelial growth factor receptor 2 (VEGFR-2), HCKproto-oncogene, Src family tyrosine kinase (HCK), fms related receptortyrosine kinase 4 (FLT4), ret proto-oncogene (RET), SRC proto-oncogene,non-receptor tyrosine kinase (SRC), platelet-derived growth factorreceptor α (PDGFR-α), platelet-derived growth factor receptor β(PDGFR-β), discoidin domain receptor tyrosine kinase 1 (DDR1),tyrosine-protein kinase KIT (KIT), colony stimulating factor 1 receptor(CSF1R), fyn related Src family tyrosine kinase (FRK), discoidin domainreceptor tyrosine kinase 2 (DDR2), lymphocyte-specific protein tyrosinekinase (LCK), LYN proto-oncogene, Src family tyrosine kinase (LYN), FYNproto-oncogene, Src family tyrosine kinase (FYN), and FGRproto-oncogene, Src family tyrosine kinase (FGR).